Liquiritin as a Tumor Suppressor Prevents the Development of Breast Cancer via the Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase 8 Signaling Pathway

Abstract

Liquiritin, a key component extracted from Glycyrrhiza radix, exhibits a variety of physiological effects. This study investigates the role of liquiritin in the progression of breast cancer. This investigation conducted experiments using two breast cancer cell lines treated with varying concentrations of liquiritin, further validating our findings in vivo. Bioinformatics analysis was used to identify the pathways potentially regulated by liquiritin in breast cancer. The results indicated that the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 8 (MAPK8) are potential downstream factors regulated by liquiritin in breast cancer. Our findings demonstrated that liquiritin significantly suppressed cell proliferation and induced cell cycle arrest in a dose-dependent manner. In addition, liquiritin triggered apoptosis by inhibiting the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B signaling pathway. Liquiritin also reduced mitochondrial membrane potential, leading to mitochondrial dysfunction and promoting excessive reactive oxygen species (ROS) production by suppressing the EGFR/MAPK8 signaling pathway. Furthermore, liquiritin treatment resulted in a notable decrease in tumor size in breast cancer models through inhibiting cell proliferation and promoting apoptosis. In conclusion, liquiritin serves as an effective tumor suppressor, suppressing the proliferation and cell cycle progression of breast cancer cells, while inducing apoptosis by regulating mitochondrial function and ROS generation via the EGFR/MAPK8 signaling pathway.

Introduction

Breast cancer is a serious life-threatening disease and is regarded as the most prevalent cancer among women worldwide (Siegel et al., 2023). The incidence and mortality rates of breast cancer remain increasing among women globally (Anastasiadi et al., 2017; Sung et al., 2021). Currently, the primary treatment approaches for breast cancer include surgery, radiotherapy, and chemotherapy, all of which can cause irreversible damage to healthy cells (Arslan et al., 2014; Shi et al., 2016). Furthermore, therapeutic strategies often involve the combination of multiple drugs or targeting various molecular pathways. Natural active ingredients derived from Chinese herbal medicine show promise in preventing breast cancer (Banik et al., 2017). Recent studies have demonstrated that certain components extracted from Chinese herbal medicine can inhibit the carcinogenesis and progression of breast cancer through the epidermal growth factor receptor (EGFR) signaling pathway (Farghadani and Naidu, 2022; Wang et al., 2019). Therefore, it is crucial to explore natural substances that are less toxic and effective against breast cancer cells.

Glycyrrhiza radix (G. radix) is a medicinal plant native to China, known for its diverse pharmacological activities, including antioxidant, anti-inflammatory, and anticancer effects (Nakatani et al., 2017; Wang et al., 2014; Yu et al., 2015). Liquiritin, a flavonoid derivative, is a main bioactive component of G. radix (Cheng et al., 2021) that helps prevent inflammatory responses and cancer progression (Dong et al., 2007; Wang et al., 2008; Yang et al., 2021). A previous study demonstrated that liquiritin induces apoptosis by suppressing mitochondrial membrane potential (MMP), induces cell cycle arrest by regulating p21, p27, cyclin B, and cyclin-dependent kinase (CDK), and triggers reactive oxygen species (ROS) production through the mitogen-activated protein kinase (MAPK)/protein kinase B (AKT) pathway in hepatoma carcinoma cells (Wang et al., 2020). The MAPK pathway generally hyperactivated in diverse types of cancers has been considered to mediate malignant phenotypes in various tumors (Ganesan et al., 2024; Low and Zhang, 2016). The activation of this pathway is an important program to promote multiple processes, such as cell proliferation, metastasis, and survival (Chen et al., 2017). In addition, liquiritin inhibits cell apoptosis by activating caspase-3 and poly ADP-ribose polymerase (PARP) (He et al., 2017). The anticancer effects of liquiritin in gastric cancer have been reported to result from its ability to promote cell apoptosis and ROS generation (Xie et al., 2017). However, the potential antitumor effects of liquiritin in breast cancer have not yet been reported.

We focused on the role and regulatory mechanisms of liquiritin in breast cancer, verifying its effects both in vivo and in vitro. Different concentrations of liquiritin were administered to MDA-MB-231 and MDA-MB-436 cells, which are commonly used to explore the effects of drugs on the development of breast cancer. We found that liquiritin exhibits anticancer effect in breast cancer. Liquiritin inhibits cell proliferation and promotes apoptosis in a dose-dependent manner. Cell cycle arrest, apoptosis, mitochondrial dysfunction, and oxidative stress were induced by liquiritin in breast cancer cells. Based on database analysis and previous literatures, we screened for downstream factors and pathways that may be regulated by liquiritin. The EGFR/MAPK8 signaling pathway was demonstrated to mediate the anticancer effects of liquiritin on breast cancer progression. Our findings provide evidence for the treatment of breast cancer with natural ingredients and open up the possibility of nonsurgical and nonchemotherapeutic approaches to alleviate breast cancer.

Methods and Materials

Data acquisition

Protein targets of liquiritin were predicted using the web tool SwissTargetPrediction (http://swisstargetprediction.ch/index.php). Genes associated with breast cancer were sourced from GeneCards (https://www.genecards.org/). The molecules potentially regulated by liquiritin in breast cancer were identified through Venn analysis and analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Pathways related to the genesis and progression of breast cancer are presented in bar graph after screening by p < 0.05 and percentage >5%.

Cell culture and treatment

Human breast cancer cell lines, MDA-MB-231 and MDA-MB-436, were incubated in L15 commercial medium (ServiceBio, Wuhan, China) supplemented with 10% fetal bovine serum (Tianhang Biotechnology, Huzhou, China).

The treatment concentrations of liquiritin (Macklin Inc., Shanghai, China) were determined using a Cell Counting Kit-8 (CCK-8) provided by Solarbio (Beijing, China). MDA-MB-231 and MDA-MB-436 cells, planted in 96-well plates, were treated with various doses (0, 25, 50, 100, 150, and 200 μM) of liquiritin for 48 h. Subsequently, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The optical density values were then measured at 450 nm using an 800 TS microplate reader (BioTek, Winooski, VT, USA). In this study, the treatment concentrations of liquiritin were designed to be approximately 1/8 IC50 (Liquiritin-L), 1/4 IC50 (Liquiritin-M), and 1/2 IC50 (Liquiritin-H). MDA-MB-231 cells were treated with 10, 20, and 40 μM of liquiritin, whereas MDA-MB-436 cells were treated with 5, 10, and 20 μM of liquiritin.

Colony formation assay

Cells (300 per well) were cultured in 60 mm plates for 14 days. Following a wash with phosphate buffer (PBS), cells were fixed with R1 reagent for 1 min. After staining with R2 reagent for 5 min, colonies were photographed using an IX53 microscope (Olympus, Tokyo, Japan).

Cell cycle detection

Cells were collected by centrifugation at 150 g for 5 min. The cell pellet was then fixed with 70% precooled ethanol at 4°C for 2 h. Subsequently, cells were incubated with RNase A for 30 min, followed by incubation with propidium iodide (PI) at 4°C for 30 min in the dark. Cell cycle distribution was then analyzed using a NovoCyte flow cytometer.

Annexin V-FITC/PI staining

Cells were collected by centrifugation, and the supernatant was removed. Cells were then resuspended in 1 mL of precooled PBS to create a cell suspension. One hundred microliter of cell suspension was mixed with 5 μL of Annexin V-FITC and incubated in the dark for 10 min. After adding 10 μL of PI in the mixture for 5 min, flow cytometry was performed immediately.

Western blot

RIPA lysis buffer (Proteintech, Wuhan, China) was used to extract proteins from the cells. Cytoplasmic and mitochondrial proteins were isolated using a mitochondrial isolation and protein extraction kit (Proteintech, Wuhan, China). The concentration of protein samples was determined using a BCA Detection Kit (Proteintech, Wuhan, China). Subsequently, sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to separate proteins at 80V for 2.5 h, followed by transferring the proteins to a polyvinylidene fluoride (PVDF) membrane (Thermo Scientific, Pittsburgh, PA, USA) at 85 V for 1.5 h. After blocking the membrane with a blocking buffer (Proteintech, Wuhan, China), the membranes were incubated at 4°C overnight with primary antibodies. Following four washes with washing buffer on a shaker, the PVDF membranes were then incubated with secondary antibodies at 37°C for 40 min. Information on all antibodies used for Western blot assay is listed in Supplementary Table S1. Proteins were visualized using enhanced chemiluminescence (Proteintech, Wuhan, China) reagents. The relative expression of proteins was quantified using blot densitometry with Gel-Pro-Analyzer software.

Hoechst staining

Cells in each group were incubated with Hoechst 33342 staining solution (Biosharp, Hefei, China) for 5 min at room temperature. Cell morphology was imaged under an IX53 fluorescence microscope.

DCFH-DA staining

Cells were harvested by centrifugation, and the supernatant was removed. The cells were incubated with 10 μM of a ROS probe (Biosharp, Hefei, China) in the dark for 30 min at 37°C. After washing by serum-free medium twice, cells were analyzed using a NovoCyte flow cytometer.

JC-1 staining

Breast cancer cells were collected and stained with JC-1 solution (Biosharp, Hefei, China) for 20 min. Following centrifugation, the supernatant was discarded. Cells were washed with JC-1 staining buffer and then resuspended in the staining buffer for analysis using flow cytometry. In addition, cells from each group were mixed with JC-1 staining solution for 20 min. After washing with JC-1 staining buffer, cells were observed under a IX53 fluorescence microscope.

Animal

BALB/c nude mice were obtained from Huachuang Sino Medical Co., LTD (Taizhou, China). A total of 12 female mice aged 6 weeks were housed under standard conditions with free access to food and water. Mice were randomly divided into two groups (n = 6) as follows: Control and Liquiritin. One week after subcutaneous injection of MDA-MB-231 cells (2 × 10⁶) into the mouse mammary fat pad, mice in the liquiritin group were administered with 30 mg/kg/day of liquiritin via intraperitoneal injection. Tumor volume was measured and recorded every 4 days. After 36 days, tumors were excised, photographed, and stored for the subsequent experiments. All animal protocols in the present study were approved by the Ethics Committee in Heilongjiang University of Chinese Medicine (No. 2024042613).

Immunohistochemistry

The collected tumor tissues were fixed, dehydrated, and embedded in paraffin. The paraffin blocks were then sectioned, and the sections were dried. After deparaffinization, sections were incubated with antigen retrieval solution at low temperature for 10 min, followed by a 15-min incubation with 3% hydrogen peroxide (Sinopharm, Shanghai, China). After blocking with bovine serum albumin, sections were incubated with anti-Ki67 (Dilution 1:200, Cat. No. 27309–1-AP, Proteintech, Wuhan, China) at 4°C overnight. Next, sections were treated with horseradish peroxidase-labeled goat anti-rabbit IgG (Dilution 1:100, Cat. No. SE134, Solarbio, Beijing, China) for 45 min. Sections were then incubated with diaminobenzidine solution (Sangon Biotech, Shanghai, China) at 37°C for 10 min. After counterstaining with hematoxylin (Solarbio, Beijing, China) for 3 min, sections were treated with ethanol hydrochloride for 3 s and immediately rinsed with running water. Finally, sections were imaged using a BX53 microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labeling assay

Following deparaffinization, tumor sections were treated with 0.1% Triton X-100 (Beyotime, Shanghai, China); terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labeling (TUNEL) staining solution (Roche, Basel, Switzerland) was then applied to the sections and incubated in the dark for 60 min. Subsequently, tissues were counterstained with 4’,6-diamidino-2-phenylindole (Aladdin, Shanghai, China) in the dark. After mounting with an antifading mounting medium (Solarbio, Beijing, China), sections were imaged using a BX53 microscope.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 8.0). Differences among groups were analyzed using a one-way analysis of variance followed by the Tukey’s multiple comparisons test. Statistical significance was defined as p < 0.05.

Results

Downstream factors potentially affected by liquiritin in breast cancer progression were screened

The chemical structure of liquiritin is depicted in Figure 1A. A total of 91 overlapping genes were identified to be associated with breast cancer and were considered potential downstream targets of liquiritin (Fig. 1B). As shown in Figure 1C, these genes were enriched in pathways implicated in tumor progression, suggesting that liquiritin may modulate breast cancer development through these pathways. The Sankey diagram illustrated key factors involved in the Ras signaling pathway, chemical carcinogenesis-ROS production, apoptosis, and metabolic pathways (Fig. 1D). Collectively, our findings suggest that liquiritin potentially regulates breast cancer progression via the EGFR/MAPK8 signaling pathway.

FIG. 1.

Downstream factors potentially affected by liquiritin in breast cancer progression were screened. (A) The structure of liquiritin. (B) Venn diagram of the molecules potentially regulated by liquiritin and related to breast cancer. (C) The overlapped genes were enriched in pathways through KEGG enrichment analysis. In the signaling pathways with p < 0.05 and percentage >5%, pathways related to the development of breast cancer are presented as a bar graph. (D) EGFR, caspase-3, caspase-8, PARP1, MAPK8, and PIK3CA are shown in the Sankey diagram. KEGG, Kyoto Encyclopedia of Genes and Genomes; EGFR, epidermal growth factor receptor; PARP1, poly ADP-ribose polymerase 1; MAPK8, mitogen-activated protein kinase 8; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Liquiritin inhibits cell cycle progression and suppresses proliferation in breast cancer cells

Based on the observed inhibition rate, we calculated the IC50 values of liquiritin in two breast cancer cell lines (Fig. 2A). Subsequently, liquiritin was used at concentrations of 10, 20, and 40 μM and 5, 10, and 20 μM for subsequent experiments, respectively. The colony formation assay demonstrated that the proliferation of cancer cells was significantly suppressed by increasing concentrations of liquiritin (Fig. 2B). Compared with the control group, liquiritin treatment resulted in a significant increase in cells in the G1 phase and a concomitant decrease in cells in the S phase (Fig. 2C). The CDK inhibitors p21 and p27 are crucial regulators of cell cycle arrest in numerous human malignancies (Abukhdeir and Park, 2008). We observed a significant upregulation of p27 and p21 expressions in cells treated with increasing doses of liquiritin. Furthermore, the expression of Cyclin B1 and CDK1 was downregulated in liquiritin-treated cells (Fig. 2D). These findings collectively confirm that liquiritin inhibits proliferation and induces cell cycle arrest in breast cancer cells in a dose-dependent manner.

FIG. 2.

Liquiritin inhibits cell cycle progression and suppresses proliferation in breast cancer cells. (A) MDA-MB-231 and MDA-MB-436 were treated with different concentrations (0, 25, 50, 100, 150, 200 μM) of liquiritin. CCK-8 was used to detect the inhabitation rate of breast cancer cells. IC50 of liquiritin in MDA-MB-231 is 79.84 μM. IC50 of liquiritin in MDA-MB-436 is 45.56 μM. (B) After treated with the low-, medium-, and high-dose of liquiritin, cell proliferation of cancer cells treated with different concentrations of liquiritin was examined by clone formation assay. MDA-MB-231 cells were treated with 10, 20, 40 μM of liquiritin, and MDA-MB-436 was treated with 5, 10, 20 μM of liquiritin. (C). The cells were stained using propidium iodide (PI) and assessed the effect of liquiritin on cell cycle using flow cytometry. (D). The protein expressions of p27, p21, cyclin B1, and CDK1 were determined by Western blot in the two breast cancer cells. n = 3. ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. CCK-8, Cell Counting Kit-8; CDK1, cyclin-dependent kinase 1.

FIG. 3.

Liquiritin induces cell apoptosis of breast cancer cells through the PI3K/AKT signaling pathway. (A) After cells were treated with different concentrations of liquiritin, they were stained by Annexin V-FITC and PI, respectively. Cell apoptosis was evaluated using flow cytometry. (B) Hoechst staining was used to present the apoptosis induced by liquiritin in different groups. Scale bar: 100 μm. (C). The alteration of cleaved PARP1, cleaved caspase-3, and cleaved caspase-8 expressions revealed the apoptosis in breast cancer cells. Western blot was used to detect their expressions to ensure the effect of liquiritin on cell apoptosis. (D and E) The phosphorylation of PI3K and AKT indicates the activation of the PI3K/AKT signaling pathway. Western blot evaluated their expressions and phosphorylation levels to confirm whether liquiritin targets the PI3K/AKT signaling pathway in MDA-MB-231 and MDA-MB-436 cells. n = 3. ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT, protein kinase B; PI, propidium iodide; PARP1, poly ADP-ribose polymerase 1.

Liquiritin induces apoptosis of breast cancer cells through the PI3K/AKT signaling pathway

Annexin V/PI staining revealed that treatment with medium and high concentrations of liquiritin (Liquiritin-M and -H, respectively) significantly enhanced the apoptosis rate, whereas low-dose liquiritin (Liquiritin-L) induced a modest increase of apoptosis in the two cell lines (Fig. 4A). Nuclei were stained with Hoechst solution, and changes in nuclear morphology and fluorescence intensity were assessed to identify apoptotic cells. We observed an increase in bright nuclear staining following liquiritin treatment (Fig. 4B), suggesting that liquiritin promotes apoptosis in breast cancer cells. Furthermore, the relative protein expression levels of cleaved PARP1, cleaved caspase-3, and cleaved caspase-8 were increased by liquiritin in both breast cancer cells (Fig. 4C). These results suggested that liquiritin activated PARP1, caspase-3, and caspase-8 to induce apoptosis. Abnormal activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling pathway is frequently observed in malignant tumors, including breast cancer (Cerma et al., 2023). Based on the KEGG enrichment analysis, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, encoding PI3K 110α, was identified as a potential downstream target of liquiritin. Liquiritin treatment reduced the protein expression of PI3K 110α in the two cell lines (Fig. 4D). Concurrently, the phosphorylation of AKT was decreased by liquiritin, indicating that liquiritin suppressed activation of the PI3K/AKT signaling pathway in breast cancer. In summary, liquiritin promotes apoptosis through upregulating PARP1, caspase-3, and caspase-8 expression and by inhibiting the PI3K/AKT signaling pathway in breast cancer cells.

FIG. 4.

Liquiritin enhances mitochondrial dysfunction and ROS generation of breast cancer cells. (A). DCFH-DA is a probe targeted ROS. Cells treated with different doses of liquiritin were incubated with DCFH-DA in the dark. The fluorescence intensity suggested the effect of liquiritin on ROS generation in breast cancer cells. (B). Cells were incubated with JC-1 probe and assessed by flow cytometry to determine the effect of liquiritin on mitochondrial membrane potential. (C). The images of fluorescence of cells stained by JC-1 in control group and Liquiritin-H group. Scale bar: 100 μm. (D). The protein expressions of cytochrome C in mitochondria and cytoplasm were examined by Western blot. n = 3. ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ROS, reactive oxygen species.

Liquiritin enhances mitochondrial dysfunction and ROS generation via the EGFR/MAPK8 signaling pathway

As shown in Figure 5A, the mean fluorescence intensity was significantly increased in the Liquiritin-M and -H groups compared with the control, which confirmed that liquiritin induced the generation of ROS. A decline in MMP is considered an early indicator of apoptosis (Marchetti et al., 1996). We observed an increase in JC-1 monomers and a decrease in aggregates in both cell lines treated with varying concentrations of liquiritin (Fig. 5B). After liquiritin treatment, the intensity of red fluorescence was reduced, whereas green fluorescence was enhanced in both breast cancer cell lines (Fig. 5C). These results indicated that liquiritin induced a reduction in MMP and promotes ROS generation. The protein expression of cytochrome C was upregulated in the cytoplasm and downregulated in the mitochondria by liquiritin, suggesting that liquiritin triggered the translocation of cytochrome C in breast cancer cells. Thus, liquiritin induces ROS production and MMP reduction, promoting apoptosis in breast cancer cells.

FIG. 5.

Liquiritin blocks the activation of EGFR/MAPK8 signaling pathway. (A) The expression and phosphorylation level of EGFR were determined in MDA-MB-231 and MDA-MB-436 cells treated with different concentrations of liquiritin. (B) The expression and phosphorylation level of MAPK8 were detected in the two cells using Western blot. n = 3. ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001. EGFR, epidermal growth factor receptor; MAPK8, mitogen-activated protein kinase 8.

EGFR activation is known to contribute to the proliferation and growth of cancer cells (Zandi et al., 2007). As shown in Figure 6A, liquiritin treatment suppressed EGFR phosphorylation in a dose-dependent manner (Fig. 6A). MAPK8 is involved in various cellular events, including apoptosis and oxidative stress. Phosphorylation of MAPK8 is required for cell apoptosis (Li et al., 2015). Following treatment with different concentrations of liquiritin, the phosphorylation of MAPK8 was decreased in cancer cells (Fig. 6B). These findings confirm that liquiritin facilitates oxidative stress and mitochondrial dysfunction by inhibiting the EGFR/MAPK8 signaling pathway.

FIG. 6.

Liquiritin inhibits tumor growth of breast cancer in vivo. (A) After subcutaneous injection of MDA-MB-231 cells into mouse mammary fat pad, mice in liquiritin group were administered with 30 mg/kg/day of liquiritin via intraperitoneal injection. The volume of breast tumors harvested from the mice was recorded every 4 days. (B) The images of tumors isolated from mice in control group and liquiritin treatment group. (C) Ki67 expression in tumor samples was examined by IHC. Scale bar: 50 μm. (D) TUNEL staining was used to reveal apoptosis affected by liquiritin in breast tumors. Scale bar: 50 μm. n = 6. ***, p < 0.001. IHC, immunohistochemistry; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labeling.

Liquiritin inhibits tumor growth of breast cancer in vivo

Liquiritin has been elucidated to inhibit proliferation and cell cycle progression in vitro and to induce ROS generation and apoptosis. Subsequently, the role of liquiritin in vivo was investigated. One week after MDA-MB-231 cell transplantation, xenograft animals were injected with 30 mg/kg/day of liquiritin for 4 weeks in vivo. Compared with the tumor volume in the control group, liquiritin treatment significantly reduced tumor volume at days 28, 32, and 36 (Fig. 7A). The morphology of tumors is presented in Figure 7B. Tumor size was remarkably decreased by liquiritin administration. In addition, liquiritin treatment suppressed Ki67 expression in tumor tissues (Fig. 7C). Images in Figure 7D demonstrated that the number of TUNEL-positive cells was increased in the tumor tissues of mice receiving liquiritin, suggesting that liquiritin induced apoptosis in vivo. In conclusion, liquiritin effectively suppressed breast cancer tumor growth by affecting both proliferation and apoptosis.

FIG. 7.

The diagram of liquiritin regulating breast cancer progression. In this study, liquiritin suppresses the cell proliferation and facilitates apoptosis through inhibiting the PI3K/AKT and EGFR/MAPK8 signaling pathways in breast cancer cells, accompanied by the downregulation of Cyclin B1 and CDK1, as well as the upregulation of p27, p21, cleaved PARP1, cleaved caspase-3, and cleaved caspase-8. PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT, protein kinase B; EGFR, epidermal growth factor receptor; MAPK8, mitogen-activated protein kinase 8; CDK1, cyclin-dependent kinase 1; PARP1, poly ADP-ribose polymerase 1.

Discussion

Despite improvements in the prognosis of breast cancer due to advancements in therapeutic approaches, the efficiency of cancer cell suppression and clearance still requires enhancement (Dastjerd et al., 2022). Numerous studies have highlighted that liquiritin, an effective and available component derived from G. radix, plays a crucial role in various cancers (He et al., 2017; Wei et al., 2017; Xie et al., 2017). Results from this study indicate that liquiritin suppresses cell proliferation and facilitates apoptosis by leading to cell cycle arrest, mitochondrial dysfunction, and ROS generation via the EGFR/MAPK8 signaling pathway in breast cancer.

The development of new drugs and therapies for cancer prevention and treatment is the key direction of global cancer research. However, the depth and breadth of basic research is still insufficient. The present study provides a new approach for the intervention and remission of tumors by validating the anticancer effect of liquiritin extracted from a natural plant. In this study, liquiritin has been confirmed to obstruct proliferation and contribute to cell cycle arrest in breast cancer via activating p27 and p21, while decreasing cyclin B1 and CDK1. A previous report has confirmed that flavonoids extracted from G. radix exhibits anti-inflammatory and anticancer effects. The flavonoids significantly inhibit the tumorigenic ability of breast cancer cell MDA-MB-231 (Jiang et al., 2018). Liquiritin, one of these flavonoids, has been indicated as a tumor suppressor to suppress the progression of breast cancer and induce apoptosis and oxidative stress in breast cancer. Our findings further validated the conclusion in the previous report. In addition, Zhou et al. have reported that liquiritin, isoliquiritin, and isoliquirigenin are crucial active components from G. radix. These components induced apoptosis through increasing p21 expression, inhibiting the AKT signaling pathway and arresting cell cycle in nonsmall lung cancer cells (Zhou and Ho, 2014). The anticancer effect of liquiritin has been demonstrated in lung cancer cells. Our study confirmed that liquiritin led to an increase in p21 and p27 and a decrease in cyclin B1 and CDK1 in breast cancer cells, as well as inhibiting cell cycle. Meanwhile, liquiritin upregulated the expressions of cleaved PARP1, cleaved caspase-3, and cleaved caspase-8, while also inducing the deactivation of the PI3K/AKT signaling pathway. Our findings further indicated that liquiritin as an effective tumor suppressor impedes proliferation and triggers apoptosis in breast cancer.

According to the results of KEGG enrichment analysis, PARP1, caspase-3, and caspase-8 in apoptosis pathway are potential downstream factors of liquiritin. Liquiritin has been reported to induce apoptosis by activating caspase-3 and PARP in human cervical cancer (He et al., 2017). The activation of caspase-3 and caspase-8 by liquiritin in gastric cancer indicates that liquiritin is an effective drug that activates apoptosis in gastric cancer cells (Xie et al., 2017). The liquiritin-induced upregulation of PARP1, caspase-3, and caspase-8 demonstrates that liquiritin significantly promotes apoptosis in breast cancer cells. Therefore, liquiritin is considered to have anticancer effects on human cancer progression. Our findings show that liquiritin suppresses PI3K 110α expression and the phosphorylation of AKT in two breast cancer cell lines, enhancing cell apoptosis via the PI3K/AKT pathway. Thus, it may serve as a tumor inhibitor with therapeutic effect on breast cancer.

Moreover, liquiritin induced mitochondrial dysfunction by upregulating ROS generation and downregulating MMP. It also inhibited the activation of cytochrome C. The activation of the EGFR/MAPK8 signaling pathway was suppressed by liquiritin, contributing to apoptosis and mitochondrial dysfunction. EGFR is generally upregulated in breast cancer and other cancers (Hagan et al., 2023). Previous studies have indicated that suppressing the EGFR signaling pathway induces cell apoptosis and mediates the anticancer effects of various drugs (Palanivel et al., 2020). The MAPK signaling pathway has been regarded as a key pathway for regulating the progression of breast cancer (Wu et al., 2024). The activation of the MAPK8 signaling pathway mediates the promotive effects of noncoding RNA molecules on the progression of colorectal cancer (Liu et al., 2022). Liquiritin has been reported to exert an antimyocardial fibrosis effect by inhibiting the MAPK8 signaling pathway (Zhang et al., 2016). It also accelerates apoptosis and induces MMP reduction by suppressing the phosphorylation of MAPK8 (Zhai et al., 2019). In addition, the MAPK pathway mediated the inhibitory effect of ononin on tumor bone metastasis in breast cancer (Ganesan et al., 2024). The activation of MAPK signaling pathway serves as an important event to support various cellular processes, including tumor growth and survival (Chen et al., 2017; Engelman, 2009; Machado et al., 2016). Our study has confirmed that liquiritin significantly suppressed the phosphorylation of the MAPK signaling pathway in a dose-dependent manner in breast cancer cells, further suggesting that the MAPK8 signaling pathway is involved in the inhibition of liquiritin on breast cancer progression. Collectively, liquiritin has been indicated to suppress cell proliferation and promote cell apoptosis and oxidative stress in breast cancer through the suppression of the EGFR/MAPK8 signaling pathway.

However, this study is limited by the fact that in vitro studies using cell lines cannot fully mimic the complex tumor microenvironment in vivo. Clinical application of the results of this study will require validation in multiple models. In the future, it is necessary to further study the anticancer properties of liquiritin and explore whether it can be combined with existing anticancer methods and adjuvant therapy to improve the poor prognosis of breast cancer. We will further contribute to the improvement of poor prognosis of breast cancer to broaden the clinical treatment options.

Conclusion

To sum up, liquiritin has been confirmed to be a tumor suppressor contributing to the improvement and therapy of breast cancer. Liquiritin inhibits proliferation through inducing cell cycle arrest in breast cancer and facilitates apoptosis through the PI3K/AKT signaling pathway. The loss of MMP and ROS generation is triggered by liquiritin, indicating that liquiritin promotes oxidative stress and mitochondrial dysfunction to suppress breast cancer development. The inhibitory effect of liquiritin on breast cancer progression was mediated by the EGFR/MAPK8 signaling pathway. Liquiritin is expected to be an anticancer candidate against the development of breast cancer.

Footnotes

Authors’ Contributions

P.L.: Conceptualization, data curation, investigation, methodology, validation, writing—original draft, and writing—review and editing. L.Y.: Data curation, investigation, and validation. Y.J.: Data curation, investigation, and validation. Y.C.: Investigation and visualization. M.Z.: Investigation and visualization. L.J.: Conceptualization, methodology, and writing—review and editing. P.G.: Conceptualization, methodology, and writing—review and editing.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author on special request.

Disclosure Statement

The authors declare that they have no competing interests.

Funding Information

This research was supported by the National Natural Science Foundation of China (No. 82304793), the Postdoctoral Foundation of Heilongjiang Province (No. LBH-Z20200), the Joint Guidance Project of Heilongjiang Natural Science Foundation (No. LH2023H072), and the Scientific Research Project of Chinese Medicine of Heilongjiang Province (No. ZHY2023-033).

Supplementary Material

Supplementary Table S1

References

Abukhdeir

, Park

. P21 and p27: Roles in carcinogenesis and drug resistance. Expert Rev Mol Med, 2008; 10:e19; doi: 10.1017/s1462399408000744

Anastasiadi

, Lianos

, Ignatiadou

, et al. Breast cancer in young women: An overview. Updates Surg, 2017; 69(3):313–317; doi: 10.1007/s13304-017-0424-1

Arslan

, Dizdar

, Altundag

. Chemotherapy and biological treatment options in breast cancer patients with brain metastasis: An update. Expert Opin Pharmacother, 2014; 15(12):1643–1658; doi: 10.1517/14656566.2014.929664

Banik

, Parasuraman

, Adhikary

, et al. Curcumin: The spicy modulator of breast carcinogenesis. J Exp Clin Cancer Res, 2017; 36(1):98; doi: 10.1186/s13046-017-0566-5

Cerma

, Piacentini

, Moscetti

, et al. Targeting PI3K/AKT/mTOR pathway in breast cancer: From biology to clinical challenges. Biomedicines, 2023; 11(1); doi: 10.3390/biomedicines11010109

Chen

, Wang

, Dai

, et al. AHNAK suppresses tumour proliferation and invasion by targeting multiple pathways in triple-negative breast cancer. J Exp Clin Cancer Res, 2017; 36(1):65; doi: 10.1186/s13046-017-0522-4

Cheng

, Zhang

, Yang

, et al. Recent advances in chemical analysis of licorice (Gan-Cao). Fitoterapia, 2021; 149:104803; doi: 10.1016/j.fitote.2020.104803

Dastjerd

, Valibeik

, Rahimi Monfared

, et al. Gene therapy: A promising approach for breast cancer treatment. Cell Biochem Funct, 2022; 40(1):28–48; doi: 10.1002/cbf.3676

Dong

, Inoue

, Zhu

, et al. Activation of rapid signaling pathways and the subsequent transcriptional regulation for the proliferation of breast cancer MCF-7 cells by the treatment with an extract of glycyrrhiza glabra root. Food Chem Toxicol, 2007; 45(12):2470–2478; doi: 10.1016/j.fct.2007.05.031

10.

Engelman

. Targeting PI3K signalling in cancer: Opportunities, challenges and limitations. Nat Rev Cancer, 2009; 9(8):550–562; doi: 10.1038/nrc2664

11.

Farghadani

, Naidu

. Curcumin as an enhancer of therapeutic efficiency of chemotherapy drugs in breast cancer. Int J Mol Sci, 2022; 23(4); doi: 10.3390/ijms23042144

12.

Ganesan

, Xu

, Wu

, et al. Ononin inhibits tumor bone metastasis and osteoclastogenesis by targeting mitogen-activated protein kinase pathway in breast cancer. Research (Wash D C), 2024; 7:0553; doi: 10.34133/research.0553

13.

Hagan

, Mander

, Joseph

, et al. Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen-induced BimEL dependent apoptosis in ER(+) breast cancer cells. Int J Oncol, 2023; 62(2); doi: 10.3892/ijo.2022.5468

14.

, Liu

, Zhou

, et al. Liquiritin (LT) exhibits suppressive effects against the growth of human cervical cancer cells through activating caspase-3 in vitro and xenograft mice in vivo . Biomed Pharmacother, 2017; 92:215–228; doi: 10.1016/j.biopha.2017.05.026

15.

Jiang

, Dai

, Pan

, et al. Total flavonoids from radix glycyrrhiza exert anti-inflammatory and antitumorigenic effects by inactivating iNOS signaling pathways. Evid Based Complement Alternat Med, 2018; 2018:6714282; doi: 10.1155/2018/6714282

16.

, Zhang

, Sun

, et al. Celastrol induces apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells: An in vitro and in vivo study. Cell Death Dis, 2015; 6(1):e1604; doi: 10.1038/cddis.2014.543

17.

Liu

, Li

, Bai

, et al. Long noncoding RNA plasmacytoma variant translocation 1 promotes progression of colorectal cancer by sponging microRNA-152-3p and regulating E2F3/MAPK8 signaling. Cancer Sci, 2022; 113(1):109–119; doi: 10.1111/cas.15113

18.

Low

, Zhang

. Regulatory roles of MAPK phosphatases in cancer. Immune Netw, 2016; 16(2):85–98; doi: 10.4110/in.2016.16.2.85

19.

Machado

, Peixoto

, Queiroz

, et al. Antiangiogenic 1-Aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas inhibit MCF-7 and MDA-MB-231 human breast cancer cell lines through PI3K/akt and MAPK/erk pathways. J Cell Biochem, 2016; 117(12):2791–2799; doi: 10.1002/jcb.25580

20.

Marchetti

, Hirsch

, Zamzami

, et al. Mitochondrial permeability transition triggers lymphocyte apoptosis. J Immunol (Baltimore, Md: 1950, 1996; 157(11):4830–4836.

21.

Nakatani

, Kobe

, Kuriya

, et al. Neuroprotective effect of liquiritin as an antioxidant via an increase in glucose-6-phosphate dehydrogenase expression on B65 neuroblastoma cells. Eur J Pharmacol, 2017; 815:381–390; doi: 10.1016/j.ejphar.2017.09.040

22.

Palanivel

, Yli-Harja

, Kandhavelu

. Alkylamino phenol derivative induces apoptosis by inhibiting egfr signaling pathway in breast cancer cells. Anticancer Agents Med Chem, 2020; 20(7):809–819; doi: 10.2174/1871520620666200213101407

23.

Shi

, Peddi

, Burton

, et al. Effect of postmastectomy radiation on survival of AJCC pN2/N3 breast cancer patients. Anticancer Res, 2016; 36(1):261–269.

24.

Siegel

, Miller

, Wagle

, et al. Cancer statistics, 2023. CA Cancer J Clin, 2023; 73(1):17–48; doi: 10.3322/caac.21763

25.

Sung

, Ferlay

, Siegel

, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021; 71(3):209–249; doi: 10.3322/caac.21660

26.

Wang

, Lu

, Liu

, et al. Liquiritigenin induces tumor cell death through mitogen-activated protein kinase- (MPAKs-) mediated pathway in hepatocellular carcinoma cells. Biomed Res Int, 2014; 2014:965316; doi: 10.1155/2014/965316

27.

Wang

, Li

, Wang

, et al. Liquiritin inhibits proliferation and induces apoptosis in HepG2 hepatocellular carcinoma cells via the ROS-mediated MAPK/AKT/NF-κB signaling pathway. Naunyn Schmiedebergs Arch Pharmacol, 2020; 393(10):1987–1999; doi: 10.1007/s00210-019-01763-7

28.

Wang

, Wang

, Tao

, et al. Curcumin derivative WZ35 inhibits tumor cell growth via ROS-YAP-JNK signaling pathway in breast cancer. J Exp Clin Cancer Res, 2019; 38(1):460; doi: 10.1186/s13046-019-1424-4

29.

Wang

, Hu

, Zhao

, et al. Antidepressant-like effects of liquiritin and isoliquiritin from glycyrrhiza uralensis in the forced swimming test and tail suspension test in mice. Prog Neuropsychopharmacol Biol Psychiatry, 2008; 32(5):1179–1184; doi: 10.1016/j.pnpbp.2007.12.021

30.

Wei

, Jiang

, Gao

, et al. Liquiritin induces apoptosis and autophagy in cisplatin (DDP)-resistant gastric cancer cells in vitro and xenograft nude mice in vivo . Int J Oncol, 2017; 51(5):1383–1394; doi: 10.3892/ijo.2017.4134

31.

, Huang

, Tian

, et al. PIWI-interacting RNA-YBX1 inhibits proliferation and metastasis by the MAPK signaling pathway via YBX1 in triple-negative breast cancer. Cell Death Discov, 2024; 10(1):7; doi: 10.1038/s41420-023-01771-w

32.

Xie

, Gao

, Yang

, et al. Combining TRAIL and liquiritin exerts synergistic effects against human gastric cancer cells and xenograft in nude mice through potentiating apoptosis and ROS generation. Biomed Pharmacother, 2017; 93:948–960; doi: 10.1016/j.biopha.2017.06.095

33.

Yang

, Dang

, Zhang

, et al. Liquiritin reduces lipopolysaccharide-aroused HaCaT cell inflammation damage via regulation of microRNA-31/MyD88. Int Immunopharmacol, 2021; 101(Pt B):108283; doi: 10.1016/j.intimp.2021.108283

34.

, Ha

, Kim

, et al. Anti-Inflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in BV2 cells and mice liver. Molecules, 2015; 20(7):13041–13054; doi: 10.3390/molecules200713041

35.

Zandi

, Larsen

, Andersen

, et al. Mechanisms for oncogenic activation of the epidermal growth factor receptor. Cell Signal, 2007; 19(10):2013–2023; doi: 10.1016/j.cellsig.2007.06.023

36.

Zhai

, Duan

, Cui

, et al. Liquiritin from glycyrrhiza uralensis attenuating rheumatoid arthritis via reducing inflammation, suppressing angiogenesis, and inhibiting MAPK signaling pathway. J Agric Food Chem, 2019; 67(10):2856–2864; doi: 10.1021/acs.jafc.9b00185

37.

Zhang

, Zhang

, et al. The protective role of liquiritin in high fructose-induced myocardial fibrosis via inhibiting NF-κB and MAPK signaling pathway. Biomed Pharmacother, 2016; 84:1337–1349; doi: 10.1016/j.biopha.2016.10.036

38.

Zhou

, Ho

. Combination of liquiritin, isoliquiritin and isoliquirigenin induce apoptotic cell death through upregulating p53 and p21 in the A549 non-small cell lung cancer cells. Oncol Rep, 2014; 31(1):298–304; doi: 10.3892/or.2013.2849

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